Neuronal BK potassium channels: Their subunit composition and distribution

K+ channels are integral membrane proteins that allow the transmembrane flux of ions through aqueous pores with high selectivity for K+ . Thus, K+ channels not only set the resting membrane potential and regulate nerve impulses; they are also essential for preventing pathological hyperactivity, such as epileptic discharges, or excessive Ca2+ influx, excitotoxicity, and cell death following brain injury. This proposal focuses on a particularly prominent and widespread type of K+ channel in the brain, the BK channel (also called the high-conductance Ca2+-activated K+ channel). The pore-forming alpha-subunit has been cloned from various mammalian species but however, very little is currently known about the subunit composition of BK channel as expressed in the brain, their respective functional contribution to the overall electrical activity of neurons, and possible association with other ion channels in synaptic microdomains. Recently a series of novel tools (e.g. selective toxins, radioligands and antibodies) became available. Using these tools, BK channels have been found pre- and postsynaptically, in a variety of important brain regions where they are located in a strategic position for dynamic feed-back control of Ca2+ influx and as a consequence, neurotransmitter release. This grant application now proposes a series of experiments to address three important questions: What is the subunit composition of BK channels in the brain? First experiments indicated that the subunit composition of neuronal BK channels is different as compared to smooth muscle. While the alpha-subunit appears to be structurally identical, a novel accessory subunit (a neuronal beta-subunit; nbeta) was detected. We plan to purify the channel complex from rat brain by a combination of chromatographic techniques as well as antibody and toxin affinity chromatography. The cDNA encoding the neuronal beta-subunit should be cloned and functionally characterized in two-electrode voltage clamp experiments using the Xenopus oocyte expression system. What is the cellular and subcellular distribution of BK channels? The distribution of the individual BK channel subunits will be investigated by high-resolution receptor autoradiography, in-situ hybridization experiments, immunocytochemical experiments and immunolgold electron microscopy. Do BK channels form functional microdomains with Ca2+ channels? The existence of macromolecular ion channel complexes will be investigated in immunoprecipitation experiments using sequence-directed antibodies against both, BK channel and Ca2+ channel subunits as well as by immunogold electron microscopy.

Ca2+-activated K+ channels with high conductance (also called BK or maxi-K channels) possess crucial functions with respect to action potential generation and -propagation in neurons as well as in muscle cells. In this respect, these ion channels serve to be an important repolarisation pathway in these tissues. In addition to the original proposal, project P12663-MED also focused in on epithilial BK channels. This new aspect to the project was initiated by the discovery that this ion channel class contributes to fluid regulation in certain epithelia (e.g. cilliary body epithelia in human eye). As the channel density is quite low in all tissues of interest, BK channels are only accessible since a few years by means of biochemical methods. This very low level of tissue expression is quite likely due to their high single channel conductance. In 1994, BK channel were biochemically purified from bovine tracheal and aortic smooth muscle and based on this purification effort, their subunit composition established. These channels are formed by association of two structurally distict subunits, alpha and beta. In this research project we focused on neuronal BK channels and could show that these channels possess a biochemically novel beta subunit, a finding which might explain their distinct pharmacological profile. The functional contribution of the beta subunit to the neuronal BK channel has not yet been established. In addition, by using sequence-directed antibodies against the pore-forming alpha subunit, we could establish the overall distribution profile of BK channels in rat brain by means of immunohistochemistry. Supplementary distribution experiments were performed using the specific radioligand [ 125 I]Iberiotoxin-D19Y/Y36F. One other important aspect of this proposal which was not mentioned in the original application was the fact that BK channels serve an important role in the regulation of aqueous humor formation in human eye. We detected this channel in the nonpigmented cilliary epithelium which tighly covers the ciliary body. This two-layered epithelium is central in the production of aqueous humor. Our functional experiments performed in rabbits indicate that BK channels serve a central role in aqueous humor production (an increased aqueous humor production and/or blocked aqueous humor outflow is responsible for one of the most important diseases of human eye, namely glaucoma). Topical application of potent nonpeptidyl BK channel blocker resulted in a significant reduction of intraocular pressure, comparable to the established glaucoma drug timolol. Noteworthy, several large drug companies initiated drug screening programs in order to search for high-affinity BK channel blockers to be introduced to glaucoma therapy.